Serotypes and Biotypes of Vibrio Cholerae O1 Isolated from Stool and Water Samples in Uzebba (Edo – State).

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Enabulele, O.I.
Faculty of Life Sciences,Department of Microbiology, University of Benin Edo State, Nigeria.

Tchounga, K.S., Ukaji, D.C., Ajugwo’ A.O.
Department of Medical Microbiology/ Medical Mycology, Faculty of Medical Laboratory Science,
Madonna University Elele Campus, Rivers State,Nigeria
All correspondence to: ukajidamian@yahoo.com

ABSTRACT
Serotype and biotype of Vibrio cholerae isolated from stool samples collected from patients with acute diarrhea and water samples were determined during September to November 2004 cholera outbreak in Uzebba in Edo–State, Nigeria. A total of 137 stool samples and 50 water samples were investigated. The samples were subjected to standard recommended microbiological techniques and confirmation of isolates by seroagglutination using Vibrio cholerae polyvalent O1 and O139 antisera and monovalent Ogawa and Inaba antisera, while biotyping was carried out by agglutination test and sensitivity to polymyxin B. Out of 137 stool samples, 63((45.98%) were found to be positive for Vibrio cholerae serogroup O1, and of 63Vibrio cholerae O1, 60(95.24%) were Vibrio cholerae serogroups O1 serotype Inaba, biotype classical and 3(4.76%) were found to be Vibrio cholerae serogroup O1, serotype Ogawa, biotype classical. Only stream water samples yielded growth of Vibrio cholerae serogroup O1, with 3(23.7%) out of 13 stream water samples. Well and bore hole water samples yielded no growth of Vibrio cholerae. Out of three Vibrio cholerae O1 isolated from stream water samples, 2(66.67%) were found to be positive, for Vibrio cholerae serogroup O1, serotype Inaba and classical biotype, while 1(33.33%) was Vibrio cholerae serogroup O1, serotype Ogawa and El -Tor biotype. This study demonstrated that some Vibrio cholerae O1 serogroup O1 isolated from stream water and stool samples had the same serotypes and biotypes. Therefore, there is need to perform a strong epidemiological surveillance for emergence and distribution of Vibrio cholerae O1.

Key words: Vibrio cholerae O1, serotype, biotype, water supplies, stool samples.

INTRODUCTION
Cholera (frequently called Asiatic cholera or epidemic cholera) is an acute diarrheal illness caused by a Gram negative slightly curved rod Vibrio cholerae (Ryan and Ray, 2004). Cholera is a worldwide problem, especially in developing countries. It has been very rare in developed nations for hundred years; however the disease is common today in other parts of the world, including the India sub-Asian continent and sub-Sahara Africa. Cholera is a major health threat in poor nations that frequently results in mortality. Although more than 100 serogroups of Vibrio cholerae exist, only two cause human disease. Vibrio cholerae O1 of which there are two biotypes (Classical and EL Tor); classified into serotypes Ogawa and Inaba and rarely Hikojima and Vibrio cholerae O139, which emerged in 1992. Cholera world wide is usually characterized by painless diarrhea and vomitting.
In 1991, there were more than a million cases in Central and South Canada. Several new cases have since been reported in Louisiana and other Gulf Coast areas (Werdlow
et al., 2002). African countries have in recent years experienced more epidemics and (WHO, 1999). A total of 29321 cholera cases and 10586 deaths were reported to WHO in 1998, with Africa accounting for the largest part with 72% of the global total (WHO, 1999). An explosive outbreak of cholera occurred among Rwanda refugees in Goma. Democratic Republic of Congo involved about 70,000 cases and caused about 12,000 deaths in 1994 (Siddique et al., 1995). In 1995, West Africa reported 64% cholera cases and 61% cases of cholera deaths (WHO, 1999). Between July and November 2001, several outbreaks were reported in the region, including 897 cases with 47 deaths in Cote d’voire and 575 cases with 21 deaths in Kano (WHO, 2001). In 2004, another outbreak involving 1316 cases and 76 deaths were again reported in Kano, while Edo-State (Uzebba) witnessed an outbreak of cholera involving 300 reported cases with 50 deaths (WHO, 2004). The Onslaught of cholera in Africa continent continues unbeaten as another one was reported in Nigeria involving 400 cases in Jigawa State, resulting in 15 deaths in October 2009 (WHO, 2009).

Strategies for prevention and control of this infectious disease depend on understanding the origin, transmission and other characteristics associated with it epidemiology. Therefore, this study was carried out to determine the prevalent serogroup, serotypes and biotypes of Vibrio cholerae responsible for the cholera outbreak in Edo-State, (Uzebba) between September to November 2004.

Materials and Methods
Stool samples were collected from patients with acute diarrihea. During the outbreak, exclusion criteria included patients using antimicrobial agents within the previous two weeks to avoid cases of antibiotic associated diarrhea. Water samples were collected in clean containers containing alkaline peptone water (APW) from various sources of water supply (streams, boreholes and wells).

Sample Processing
Stool samples collected in plain containers were cultured directly onto Thiosulphate Citrate Bile Salt Sucrose (TCBS) agar plates and incubated at 37oc for 24 hours. Water samples inoculated in APW were incubated at 37oc for 4 to 6 hours, after the incubation period, from the inoculated APW, plating was done onto TCBS agar and the plates incubated at 37oc for 24 hours. The isolates were purified by sub-culturing single colonies on sterile Nutrient agar plates which were then incubated at 37oc for 24 hours. Following standard morphological and biochemical tests according to Buchanan and Gibbons (1974), characteristic colonies grown on the selective TCBS agar were then confirmed for identification. The series of biochemical tests commonly used to identify V. cholerae (Baumann and Schubert, 1984; West and Cowell, 1984) were originally designed for clinical samples in order to specifically detect pathogenic vibrios. The series of biochemical tests include: Gram staining, oxidase , glucose and L-arabinose , methyl red , voges-proskauer and ornithine tests were performed.

Serological Test
Serological identification of isolates was done using slide agglutination test (Sakazaki and Donovan, 1984; Shimada et al., 1994), using polyvalent Vibrio cholerae O1 and Vibrio cholerae O139 antisera and monovalent Ogawa and Inaba antisera.

Biotyping
Biotyping of Vibrio cholerae isolates was done by using agglutination with chicken red blood cells and polymixin in B (50 units) sensitivity test (WHO, 1987).

RESULTS
A total of 137 stool samples collected from hospitalized patients during cholera outbreak from September to November 2004 in Uzebba (Edo – State), and water samples 50(13 stream water, 20 borehole water and 17 well water samples) were studied.

Table 1 shows the frequency of isolation, serotyping and distribution of Vibrio cholerae. Isolated from stool and water samples in Uzebba. Out of 137 stool samples, 63(45.98%) were found positive for Vibrio cholerae O1 and 60(95, 24%) out of 63, belonged to Inaba serotype, while 3(4.76%) were Ogawa serotype. None of the samples yielded growth of Vibrio cholerae O139. Water samples were also analyzed bacteriologically, 3((23.07%) out of 13 water samples collected from streams yielded growth of Vibrio cholerae O1 and borehole and well water samples yielded no growth. Table 2 shows the distribution of Vibrio cholerae O1 biotype isolated from stool and water samples. All vibrio cholerae O1 from stool samples both Inaba and Ogawa serotypes were Classical biotype, two Vibrio cholerae O1 Inaba serotype isolated from stream water samples were Classical biotype, while only Vibrio cholerae O1 Ogawa isolated from stream water sample was EL Tor biotype.
DISCUSSION
Cholera continues to be an important pubic Health problem among many poorer and vulnerable communities despite the fact that bacteriology, epidemiology and public health aspects of the disease were described in detail over a centuary (Shears, 2001). In the study, we isolated and identified 66 Vibrio cholerae out of which 63 isolates were from stool samples and 3 isolates from water samples.It was observed after serological test that the isolates were Vibrio cholerae O1 and that there was no serogroup O139 isolates. These findings are in agreement with the report of Tamang et al.,( 2005) reported that Vibrio cholerae O1 were predominant in Nepal, also similar reports were presented by Urassa et al.,( 2000);Inaet al.,(2007) reported that during his study in Manhica District hospital Southern Mozambique, all isolates were Vibrio cholerae O1. During this study, it was noticed that two serotypes Inaba and Ogawa co-existed with Vibrio cholerae O1 serotype Inaba being the most predominant with 62(93.94%) while 4(6.06%) were Vibrio cholerae O1 Ogawa serotype; and these isolates were both from stool and water samples. Our findings are in accordance with reports of Mercy et al.,( 2004) who isolated Vibrio cholerae O1 in Ghana with Inaba serotype having the highest occurrence over Ogawa serotype. Similar report was presented by Shukla et al., ( 2006 ) who recorded an emerged predominance of Vibrio cholerae O1 serotype Inaba over Ogawa between 2004 – 2006 in East Delhi.

The co-existence of the two serotypes recorded during our study was not in conformity with Inacio et al., (2007) who reported that only Vibrio cholerae O1 serotype Ogawa was isolated in Manhica District hospital Southern Mozambique. Our findings also contradicted claims by Hossein et al., ( 2005) that after a six year study on Vibrio cholerae in South Eastern Tran, all Vibrio cholerae O1 isolated were Ogawa serotype. EL Tor was the predominant biotype causing outbreak up till 1998. But during our study in Uzebba, the Classical biotype emerged predominant that although that one of the isolates from water sample was EL Tor biotype. The high emergence of Classical biotype in Uzebba could be justified by the
reports of Rafi et al., ( 2004) also isolated Vibrio cholerae O1 Classical biotype between 2000 – 2001 in Rawalpindi. This high occurrence of Vibrio cholerae O1 Inaba and Classical biotype in our study is not in accordance with the reports of Bradley et al.,(1997), Jacques et al.,( 2002) and Iwanaga et al.,( 2004), who reported that most of the strains of Vibrio cholerae O1 isolated from Madagascar, Bangladesh, Tanzania, Zaire, Latin America, Southern and Eastern regions of India were Ogawa serotype and EL Tor biotype. Our study revealed that high percentage of Vibrio cholerae isolated from both stool and water samples were Vibrio cholerae O1 Inaba and Classical biotype, which is also contrary to the findings of Hossein, et al.,(2005) that all strains of Vibrio cholerae O1 isolated between July and September 1998 in Goa belonged to EL Tor biotype, 53(66%) of them being Ogawa serotype, while 21(26%) were Inaba serotype.

Some isolates obtained from stream water samples when serotype and biotype were reported as Vibrio cholerae O1 Inaba serotype Classical biotype same as some of the isolates obtained from patient’s stool samples during the outbreak. One could be tempted to say that by referring to the phenotypic and genotypic characteristics of the isolates, this stream water that has been one of the sources of water supply in Uzebba community must have played an important role in the spread of cholera outbreak in that region. We can also say that outbreak in Uzebba is defined by social and environmental factors.The importance of aquatic reservoir depends on sanitary conditions of the community(CEDECO,2010. Cholera is spread mainly through drinking fecal – contaminated water. . While contaminated water remains the major route for cholera transmission (Shapiro, et al., 1999) food and utensils are also important (Rabbani and Greenough, 1999), emphasizing the importance of hygiene within the house hold (Fotedal, 2001).

Effective food hygiene measures include cooking food thoroughly and eating it while still hot; preventing cooked foods from being contaminated through contact with raw food or drinking water is important (Seas and Gotuzzo, ensuring proper management of excreta to avoid contamination of other water sources were important measures to reduce cholera transmission.   Education of the population at risk regarding appropriate hygienic practice is always recommended. Identification of local customs that place people at risk is also important in order to eliminate such practices (Sears and Gotuzzo, 2000). A greater understanding of the pathogen, its biology, ecology, epidemiology and strategies for treatment and prevention are essential to guide policies and programmes for the control of cholera. . Adequate measures to improve hygiene and sanitation and supply of safe potable water are needed to prevent any future outbreak of cholera in Uzebba.

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